This year we produced 5 biobricks each of which produce ssDNA which can come together to form a functional tetrahedron structure in presence of its target. The inserts were ordered as gBlocks fragments from Integrated DNA technologies As shown in the diagram, the insert can be divided into 5 parts:
- Biobrick prefix and suffix
- ProD - This is the Promoter for our biobrick. The promoter allows for high expression of our downstream product
- ssDNA sequence: This sequence is complementary to the ncRNA sequence that will be produced.
- HTBS: HIV reverse transcriptase binding site
- BBa_B0054 : Strong Terminator
We expect that upon transcription a ncRNA will be produced with an HTBS sequence that will allow HIV reverse transcriptase to bind the ncRNA and produce the desired DNA sequence. Upon addition of RNase H and RNase A the DNA: RNA duplex and the degradation can occur respectively.
As shown before the Pre-tetra and the Tetra is generated using five nucleotide sequence and we have made a total of 5 biobricks, one biobrick for each.
Figure 1: Structure of insert
Figure 2: Production of biobrick
Figure 3: Polyacrylamide gel electrophoresis (PAGE) of individual oligonucleotides of our DNA nanostructure